tbuckley/cabot-search huggingface dataset:
Abstract P2031: Myofibroblast-specific Smad7 Attenuates Fibrosis Of The Pressure-overloaded Heart By Inhibiting Mmp2-mediated Collagen Denaturation And By Restraining Macrophage Activation.
Authors: Claudio Humeres, Silvia C Hernandez-Velasco, Anis Hanna, Izabela Tuleta, Harikrishnan Venugopal, Akihiko Kubota, Nikolaos Frangogiannis
Journal: Circulation Research (2023)

Expansion and activation of cardiac fibroblasts contributes to adverse remodeling, fibrosis and dysfunction in the pressure-overloaded heart. Although early fibroblast TGF-β/Smad3 activation protects the pressure overloaded heart by preserving the matrix, sustained TGF-β activation may be deleterious, accentuating fibrosis and dysfunction. We hypothesized that induction of the endogenous TGF-β inhibitory Smad, Smad7, may attenuate fibroblast activation and deposition of collagen, limiting myocardial fibrosis and dysfunction in the pressure-overloaded heart. In a mouse model of pressure overload induced through transverse aortic constriction (TAC), Smad7 was upregulated in cardiac fibroblasts and myofibroblasts. Mice with myofibroblast-specific loss of Smad7 had increased mortality, systolic dysfunction, dilative remodeling and accelerated diastolic dysfunction in response to TAC. Moreover, myofibroblast-specific Smad7 loss hearts had higher collagen deposition and denaturation, as well as increased deposition of the fibrogenic protein periostin. Secretomic analysis showed that fibroblast Smad7 loss accentuates secretion of matrix metalloproteinase-2 (MMP2), extracellular matrix proteins and structural collagens. In vitro, the antifibrotic effects of Smad7 on fibroblast-induced collagen denaturation and pad contraction were partly mediated via MMP2 downregulation. Ingenuity pathway analysis predicted the fibrogenic matricellular protein CCN2/CTGF to be activated in Smad7 null fibroblasts. Surprisingly, myofibroblast-specific Smad7 loss increased myocardial macrophage infiltration in the pressure-overloaded heart. In vitro experiments suggested paracrine effects of Smad7 KO fibroblasts on macrophage proliferation and fibrogenic activation that involved the fibroblast-secreted mediators CD5L, SPARC, CTGF and TGFBI. In summary, the anti-fibrotic effects of Smad7 in the pressure-overloaded heart involve not only reduction in collagen deposition, but also suppression of MMP2-mediated matrix denaturation and paracrine effects inducing fibrogenic activation of macrophages.
Abstract P3206: Sex-specific Differences In Proteasome Activity And Mitochondrial Function In Mouse Hearts Lacking Natriuretic Peptide Receptor A
Authors: Shuchita Tiwari, Abhishek Mishra, Kishore Pasumarthi
Journal: Circulation Research (2023)

Objective: Atrial natriuretic peptide (ANP) is a cardiac hormone that plays an important role in cardiovascular function and energy metabolism by signaling through natriuretic peptide receptor A (NPRA). Genetic ablation of NPRA in mice resulted in hypertension, cardiac hypertrophy, and fibrosis. The cardiovascular phenotypes were reported to be more severe in male NPRA-knockout (KO) mice. It is known that reductions in proteasomal activities can lead to mitochondrial dysfunction. Similarly, an imbalance between mitochondrial fission and fusion events can impair mitochondrial function. We hypothesized that alterations in proteasomal function and mitochondrial dynamics might form the basis for phenotype differences reported for NPRA-KO mice. Method/Results: Non-invasive echocardiography, RT-QPCR, Western blotting, and proteasomal assays were performed to evaluate cardiac functions. Preliminary results revealed significant decreases in the activities of two proteasome subunits (β1 and β5) in male and female NPRA-KO hearts compared to sex-matched wild-type (WT) hearts. Notably, the percent-activity reductions were much higher in male NPRA-KO hearts. Western blotting analysis revealed that the expression levels of mitochondrial fusion proteins (Mfn1, Mfn2, and Opa1) were significantly increased in both male and female NPRA-KO hearts compared to WT controls. However, the expression of fission proteins (Drp1 &amp; MFF) was significantly increased only in male NPRA-KO hearts. Consistent with a more imbalance in mitochondrial dynamics in male NPRA-KO hearts, the expression levels of enzymes that promote fatty acid oxidation (ECHS1 and phospho-PDH) were unfavorably altered in male but not in female NPRA-KO hearts compared to controls. In addition, various echocardiographic and electrocardiographic parameters related to the cardiac function such as cardiac output, diastolic volume, left ventricular wall thickness, QRS duration, and stroke volume, were altered significantly more in male than in female NPRA-KO hearts. Conclusion: Our findings suggest that more pronounced sex-specific alterations in proteasome/mitochondrial functions in male NPRA-KO hearts can lead to changes in cardiac metabolism and a severe disease phenotype.
Abstract P1178: APOE-NOTCH Axis Governs Elastogenesis During Human Cardiac Valve Remodeling
Authors: Ziyi Liu, Yu Liu, Zhiyun Yu, Nicole Pek, Anna O'Donnell, Ian Glass, Minzhe Guo, Ya-Wen Chen, Joseph Wu, Katherine E Yutzey, Yifei Miao, Mingxia Gu
Journal: Circulation Research (2023)

Background: Valve remodeling is a complex process involving extracellular matrix organization, development of trilaminar structures, and physical elongation of valve leaflets. However, the cellular and molecular mechanisms regulating valve remodeling and their roles in congenital valve disorders remain poorly understood. Methods: Semilunar valves and atrioventricular valves from healthy and age-matched human fetal hearts with pulmonary stenosis (PS) were collected. Single-Cell RNA-sequencing (scRNA-seq) was performed to determine the transcriptomic landscape of multiple valvular cell subtypes in valve remodeling and disease. Results: scRNA-seq analysis of healthy human fetal valves identified a novel APOE+ elastin-producing valve interstitial cell (Elastin-VIC) subtype spatially located underneath VECs in the direction of unidirectional flow. Knockdown of APOE in fetal VICs resulted in significant elastogenesis defects. In pulmonary valve with PS, we observed decreased expression of APOE and other genes regulating elastogenesis such as EMILIN1 and LOXL1 , as well as elastin fragmentation. Cell-cell interaction analysis revealed that JAG1 from unidirectional VECs activates NOTCH signaling in Elastin-VICs through NOTCH3. In vitro Jag1 treatment in VICs increased the expression of elastogenesis-related genes and enhanced contractile functions with related gene expression. This was accompanied by activation of NOTCH signaling and elastogenesis observed in VICs co-cultured with VECs in the presence of unidirectional flow. Notably, we found that the JAG1-NOTCH3 signaling pair was drastically reduced in the PS valves. Lastly, we demonstrated that APOE is indispensable for JAG1-induced NOTCH activation in VICs, reinforcing the presence of a synergistic intrinsic and external regulatory network involving APOE and NOTCH signaling that is responsible for regulating elastogenesis during human valve remodeling. Conclusion: scRNA-seq analysis of human fetal valves identified a novel Elastin-VIC subpopulation, and revealed mechanism of APOE and NOTCH signaling mediated elastogenesis during valve remodeling, which provided opportunity to understand pathogenesis of elastic malformation within congenital valve disease.
Abstract P3158: Extracellular Vesicles From Ischemic Myocardium Mediate Regulatory T Cell Dysfunction Through MicroRNA-1-mediated Suppression Of VEGF-A
Authors: Zahra Mohammadigoldar, Ke Liao, Ahmed G Ibrahim, Liang Li, Weixin Liu, Eduardo Marban
Journal: Circulation Research (2023)

Motivation: Myocardial infarction (MI) triggers an efflux of pro-inflammatory extracellular vesicles (EVs) from the heart into the circulation. Here, we test the hypothesis that circulating ischemic heart EVs (iEVs) attenuate regulatory T cell (Treg) proliferation and function post-MI. Methods: Hearts were explanted from mice and Langendorff-perfused with Tyrode buffer either continuously for two hours (nonischemic), or subjected to no-flow for 30 min, and reperfused for 1.5h. Heart effluent (~700ml) was collected for EV isolation by ultracentrifugation. EV size and concentration were measured by nanoparticle tracking analysis; EV markers were characterized by western blot. EVs from nonischemic heart effluent (nEVs) served as controls. RNA sequencing (RNAseq) followed by qPCR validation was used to identify microRNAs in EVs. Assessments of human Tregs included cell proliferation (Cell Counting Kit-8 assay), IL-10 production (qPCR of Tregs, and ELISA of Treg-conditioned media), and RNAseq after exposure to EVs. In vivo, an antagomir was administered retro-orbitally immediately post-MI, and splenic Tregs were measured by flow cytometry 7 days post-MI. Findings: MiR-1-3p was highly enriched in iEVs compared to nEVs. Exposure of human Tregs to iEVs or a miR-1 mimic resulted in decreased proliferation ( P &lt;0.0001, N=8) and production of IL-10 (qPCR: P&lt;0.0001, n=9; ELISA: P &lt;0.0001, N=6) compared to nEVs. Antagomir-1 reversed iEV- and miR-1-induced inhibition of Treg proliferation. RNAseq on human Tregs revealed that VEGF-A, a miR-1 target gene, was suppressed after exposure to iEVs compared to nEVs. In vivo, the number of splenic Tregs decreased on day 7 post-MI, with a concomitant decrease in IL-10 + and VEGF-A + Tregs. Intriguingly, administration of antagomiR-1 restored numbers of splenic IL10 + and VEGF-A + Treg cells. Conclusions: iEVs cause Treg dysfunction by transferring miR-1. In turn, miR-1 suppresses VEGF-A, a known modulator of Treg function. Our findings reveal novel paracrine modulation of immune cells by ischemic myocardium. Targeting EV signaling post-MI represents a potential new strategy to augment Treg-mediated tissue repair.
Abstract P3037: MicroRNA-205 Is Required For Normal Cell Cycle And Cardiac Growth In The Neonatal Mouse Heart.
Authors: Patrick G Burgon, Jonathon Weldrick, Rui Yi, Lynn Megeney
Journal: Circulation Research (2023)

After birth, most newborn mouse cardiomyocytes (CM) undergo a final cycle of cell division in the absence of cytokinesis, which leads to binucleation. The majority of subsequent increases in cardiac mass are attributable to cardiomyocyte hypertrophy, as a negligible number of new CM are created after birth. Mice with a heart-specific deletion of Dicer, a crucial enzyme necessary for microRNA maturation, die by the fifth postnatal day in spite of having normal cardiac morphology and function at birth. Despite the significance of this phenomenon, little is known about the molecular/genetic foundation for the change from hyperplastic to hypertrophic-based myocardial expansion that occurs in the neonatal mouse heart. We hypothesize that a microRNA is required for the normal transition from fetal to adult heart during the neonatal period. Heart microRNA profiles (3 arrays/time point) were measured from RNA was isolated from E19, 1, 3, 5, 7, 10, and 35-day-old mouse hearts (9 hearts/time point pooled). Bioinformatic analysis identified microRNA-205 as a possible participant in the cardiac transitional program. We observed a transient 20-fold increase in miR205 expression between postnatal days 1 and 5, with levels returning to baseline by day 10. In-situ hybridization revealed miR-205 expression is restricted to the heart's epicardium.We next generated mice with a CM-specific deletion of miR205 using MHCcre targeting of a floxed-miR205 allele. Mice lacking miR-205 in their CM appear normal at birth. Elevated cell cycle markers (Ki67 &amp; phospho-pH3) were detected at postnatal day-14 with 20% larger hearts at postnatal day 14, and adult hearts measuring up to 50% larger than controls. In contrast, a CM-specific miR205 over-expression mouse model resulted in more CM being present at both 5-day and 14-day post-birth. Interestingly, no difference in cell-cycle markers or heart size was observed. miR205 directly targets YAP within the evolutionarily conserved hippo pathway, which regulates organ size. YAP protein expression is significantly increased in hearts lacking miR205. Both of the miR205-heart models present with altered Hippo signaling kinetics after birth. We conclude that miR205 regulates postnatal heart size by directly modulating the Hippo pathway.
Abstract P2068: β <sub>Iv</sub> -spectrin/stat3 Are Responsible For Concentric Versus Eccentric Cardiac Hypertrophy
Authors: Drew Nassal, Shivangi Mohta, Nehal Patel, Xianyao xu, Mei Han, Arnav Divekar, Nicholas Leahy, Peter J Mohler, Thomas J Hund
Journal: Circulation Research (2023)

Hypertrophy is critical to development of heart failure following cardiac stress. In pressure overload, hypertrophy is divided by early concentric and chronic eccentric orientations, however, the mechanisms regulating this transition and the impact on heart function are largely unknown. Previous work identified the structural protein β IV -spectrin and its interaction with the transcription factor, STAT3, in coordinating hypertrophic profiles. Specifically, mice expressing truncated β IV -spectrin lacking interaction with the kinase CaMKII ( qv 3J mice) resisted β IV -spectrin degradation and maintained concentric hypertrophy and systolic function compared to WT following 6 weeks transaortic constriction (TAC). Alternatively, mutant β IV -spectrin mice lacking the β IV -spectrin/STAT3 binding domain ( qv 4J ) expressed reduced EF and LV dilation, consistent with eccentric remodeling, suggesting β IV -spectrin/STAT3 is a regulator of cardiac profiles. This relationship was examined by adenoviral expression of the hypertrophic driver MEK1/2 on isolated myocytes, where MEK increased the width of WT cells but length in qv 4J . Notably, application of STAT3 inhibitor S3I-201 on qv 4J myocytes restored growth in width. Additionally, in vivo delivery of S3I-201 to WT mice after 6 weeks TAC reduced LV dilation and recovered systolic function. To enable hypertrophy, recent work identified the need for microtubules to traffic and locally translate sarcomeric transcripts. Therefore, we tested the hypothesis that β IV -spectrin/STAT3 direct orientation by altering microtubule stability and sarcomeric transcript trafficking. Isolated myocytes from WT and qv 4J mice were treated with the microtubule depolymerizing drug Nocodazole and then washed to evaluate microtubule repolymerization. Microtubules restored faster in qv 4J myocytes but could be reduced to WT levels by treating with S3I-201. Furthermore, localization of the sarcomeric gene, actc1 (α-actinin) by RNAScope showed more peripheral localization in qv 4J myocytes, compared to WT, and was normalized by S3I-201. Together, these data identify β IV -spectrin/STAT3 as modulators of microtubule dynamics, sarcomeric transcript distribution, hypertrophic orientation, and systolic function.
Abstract P2111: Characterizing Human Cardiac Fibroblast Responsiveness To Hemodynamic Unloading In Heart Failure With Reduced Ejection Fraction
Authors: Rachel Biggs, Daniel N Silverman, Yuhua Zhang, Catalin F Baicu, Michael R Zile, Amy Bradshaw
Journal: Circulation Research (2023)

Background: Myocardial interstitial fibrosis is a common pathology in cardiomyopathies leading to ventricular dilation and increased hemodynamic load that result in heart failure with reduced ejection fraction (HFrEF). Previous research has shown that HFrEF patients treated with a left ventricular assist device (LVAD) undergo hemodynamic unloading resulting in at least partial cardiomyocyte recovery. However, evidence supports that these patients do not experience a regression of fibrosis and demonstrate, in some cases, a worsening of fibrosis after LVAD treatment. Purpose: We hypothesize that human cardiac fibroblasts (HCFs) are constitutively activated in HFrEF myocardium but remain unresponsive to hemodynamic unloading with LVAD placement. Methods and Results: Forty human subjects with HFrEF undergoing LVAD implantation were enrolled to provide a portion of myocardium routinely removed during LVAD placement. In addition, 7 biopsies previously collected from transplanted hearts with extended LVAD treatment were also evaluated (LVEX). Quantification of PSR stained sections revealed a significant increase in collagen content in the HFrEF tissue (Collagen volume fraction % (CVF) = 2.8±0.2) in comparison to control tissues (CVF = 0.9±0.2) that remained elevated in LVEX hearts (CVF = 3.1±0.3). HCFs derived from biopsies received at LVAD placement were isolated and grown to confluence. HCFs from HFrEF patients and control HCFs from healthy donors were then plated on substrates with mechanical stiffnesses reflective of either normal myocardium (2kPa) or failing myocardium (8kPa). Cells were collected at 4- and 7-day timepoints and levels of collagen I (Col I) and alpha-smooth muscle actin (α-SMA) were quantified through western blot analysis with β-actin as a loading control. Whereas control HCFs were responsive to changes in substrate stiffness producing more Col I and α-SMA on 8kPa versus 2kPa, HCFs from HFrEF patients were unresponsive to changes in stiffness exhibiting no significant difference in production on 2 vs. 8kPa. Conclusion: These data suggest that HCFs isolated from the failing myocardium do not respond to changes in mechanical load and hence, might contribute to persistent increases in fibrosis in failing and unloaded hearts.
Abstract P3087: Macrophage Fatty Acid Metabolism Promotes Splenic Hematopoiesis Through Mitochondrial Signaling During Heart Failure With Preserved Ejection Fraction
Authors: Mallory Filipp, Zhi-Dong Ge, Connor Lantz, Sanjiv J Shah, Edward B Thorp
Journal: Circulation Research (2023)

Rationale: Diastolic dysfunction, including during heart failure with preserved ejection fraction (HFpEF), is a widespread morbidity with limited therapeutic options. Though heterogeneous in etiology, major risk factors include hyperlipidemia, hypertension, and inflammation. Recent studies have also shown significant changes in systemic metabolite availability during HFpEF, further suggesting that metabolic changes may be an important indicator of HFpEF pathophysiology. Macrophages, which have previously been implicated in the promotion of diastolic dysfunction, utilize specific metabolic pathways to drive inflammation or tissue repair. However, little is understood about myeloid metabolism during HFpEF. Objective: We set out to define the functional and metabolic heterogeneity of the cardiac immune response during diastolic dysfunction. Methods/Results: Diastolic dysfunction was induced by HFD and eNOS inhibition in mice. scRNAseq showed a specific induction of pro-fibrotic and lipid handling pathways in cardiac macrophages, including the fatty acid receptor Cd36. In live animals, myeloid deficiency of Cd36 prevented diastolic dysfunction and was associated with dampened cardiac inflammation. Investigating immune cell sourcing during HFpEF revealed that a splenic hematopoietic niche formed in wild-type mice. Further, mitochondrial metabolism in splenic macrophages was associated with increased expression of VCAM-1, the latter of which has been shown to play a role in retention of HSCs in the spleen. Indeed, blocking VCAM-1 or genetic deletion of VCAM-1 in myeloid cells was sufficient to disrupt retention of HSCs within the spleen during HFpEF. Culturing splenic macrophages with saturated fatty acids (SFAs) in vitro was sufficient to induce expression of Vcam1. Increased cellular citrate levels were correlated with increased histone acetylation at the Vcam1 promoter in splenic macrophages cultured with SFAs. Inhibition of mitochondrial uptake of fatty acids through CPT1a siRNA or inhibition of ACLY were sufficient to prevent this upregulation of Vcam1 expression. Conclusions: Our data suggest a critical role for myeloid metabolism in the sourcing of detrimental-monocyte-derived macrophages from the spleen to the heart.
Abstract P3084: Neutrophil Expansion And Netosis Are Essential For Adverse Left Ventricular Remodeling And Dysfunction In Chronic Ischemic Heart Failure
Authors: Gregg Rokosh, Sergey Antipenko, Nicolas Mayfield, Miki Jinno, Matthias Gunzer, Ameen Ismahil, Tariq Hamid, Sumanth D Prabhu
Journal: Circulation Research (2023)

Chronic inflammation contributes significantly to disease progression in heart failure (HF) with the expansion and adverse activation of macrophages, dendritic cells, and T cells in the failing heart. Neutrophils play a well-known role in the initial acute immune response to myocardial infarction (MI), however, whether and how they contribute to inflammation and adverse LV function and remodeling in chronic ischemic HF is unknown. We tested the hypothesis that activated neutrophils are required for tissue remodeling and disease progression in HF in mice studied 4 and 8 weeks after MI during established ischemic HF (vs sham). CD11b + Ly6G + neutrophils were expanded in peripheral blood (PB), heart, spleen and bone marrow 8 w post-MI. Neutrophils in the failing heart were localized to the MI border zone and augmented in remote zone myocardium by immunofluorescent staining. CXCL1 and CXCL5 expression was increased in the failing heart, consistent with chemotactic signals for myocardial neutrophil recruitment. Plasma from HF mice elicited greater chemotaxis in naïve neutrophils (vs naïve plasma) by time-lapse video microscopy. Further, PB neutrophils from HF mice exhibited significantly increased neutrophil extracellular trap (NET) production, with concomitant increase in plasma NETs (histone-DNA complexes) in HF mice. The direct pathological impact of NETs was demonstrated by intramyocardial injection of isolated NETs into naïve hearts mimicking HF with decreased EF, LV dilation, and fibrosis. Finally, the in vivo role of neutrophils in chronic HF (4 and 8 w post-MI) was determined via depletion studies using genetic (Ly6G-diptheria toxin receptor mice) and antibody (1A8) based approaches. Neutrophil depletion significantly improved LV EF and reduced end systolic and diastolic volumes. 1A8 treated HF mice also exhibited reduced remote zone fibrosis. The necessity of NETosis in HF development was confirmed using neutrophil and myeloid – specific PAD4 deletion where the decline in LV EF and increase in volumes in HF were attenuated. In conclusion , neutrophil expansion and NETosis are important contributors to chronic inflammation in HF and play an obligatory role in the development of LV dysfunction and remodeling in ischemic cardiomyopathy.